The purpose of this project is to relate structural features of the ADV genome to the ability of various virus isolates to replicate in culture and to cause disease in mink. We have continued to study molecular clones chimeric for genomic segments between the nonpathogenic ADV-G and pathogenic ADV strains. By performing segmental exchanges and doing PCR mutagenesis, we have found that mutating a single glycine residue to aspartic acid in the capsid proteins gene abrogates the ability of an infectious ADV-G clone to replicate and produce infectious virus in cell culture. By comparing transfections with full length clones, we have found that the block to replication appears to be a failure to generate single stranded progeny DNA. None of the viruses rescued from full length clones cause disease in adult mink, suggesting that pathogenicity of ADV and its ability to replicate permissively in CRFK cells are linked in a negative fashion. Additional ADV-Utah 1 sequence information has been obtained which indicates substantial sequence variation not previously recognized. Using prokaryotic expression systems, we demonstrated the existence of a nonstructural protein-2 (NS-2) for ADV, but have failed to identify a predicted gene product of the ADV RX mRNA (NS-3). Recombinant vaccinia viruses expressing either both VP1 and VP2 or VP2 singly synthesize appropriately sized proteins; the proteins are transported to the nucleus and form empty virus particles. Thus VP1 is not required for particle assembly. The VP1/VP2 capsids, but not the VP2 only ones, can bind to ADV DNA replicative forms like bona fide ADV virions. The recombinant viruses stimulate anti-ADV antibody in mice, but appear to be poorly immunogenic in mink. A similar VP1 and VP2 segment has been introduced into a recombinant baculovirus, denoted AcADV, which also expresses both proteins and the proteins self-assemble into capsids.